Cardiac disease is the leading cause of death in the United States, largely due to the inability of the heart to repair itself following injury. Recent therapies for heart disease have investigated the potential of stem cells to enhance cardiac repair and regeneration. In particular, mesenchymal stem cells (MSCs) have shown beneficial effects in vitro, in vivo and in clinical trials. While the results from this work are exciting, th mechanism by which MSCs enhance cardiomyocyte function remains unknown. Understanding how MSCs are mediating their beneficial effect is key to developing safer and more effective therapeutics. One challenge in studying MSC biology is the lack of a controlled in vitro environment that permits interrogation of key mechanistic components of MSC biology yet maintains high biofidelity for clinical relevance. Cardiac tissue engineering provides a novel approach to studying MSC biology since the culture environment can be tightly controlled yet the basic three-dimensional architecture of natural myocardium is maintained. Using an engineered cardiac tissue (ECT)-based approach with neonatal rat cells, we have previously shown that MSC- supplementation to ECTs enhances their function. The observed beneficial effects may reflect one or both of the following: 1) that MSCs are having an intrinsic effect on the tissue function via direct cell-cell coupling; 2) the MSCs are having an extrinsic effect on myocyte function via the release of beneficial paracrine factors. The aims of this F30 resubmission proposal are to investigate the relative contributions of 1) intrinsic and 2) extrinsi effects of MSCs to the enhancement of cardiomyocyte function. To increase clinical relevance, all proposed studies exclusively utilize cardiomyocytes derived from human embryonic stem cells (hCMs), and human MSCs (hMSCs). Three tissue types will be created: hCM-only, hCM-hMSC hybrid, and hMSC-only. Intrinsic effects will be examined via immunohistochemical analysis of the intercalated disk proteins connexin- 43 and N-cadherin in the hybrid tissues. Following identification of cell junctions, shRNA to both proteins will be transfected into MSCs prior to tissue construction to inhibit cell-cell connection. Contractile and electrical function o the tissues will then be assessed to determine the contribution of direct cell contact in mediating the beneficial MSC effects. Additionally, the ability of MSCs to align cells within the hECT will be assessed before and after shRNA transfection. Extrinsic effects will be examined using a novel side-by-side culture system of myocyte-only tissues with either the hybrid or MSC-only tissues to isolate paracrine effects without direct cell contact. Biochemical and proteomic analysis of conditioned media from either hybrid or MSC-only tissues will be performed to identify factors responsible for mediating the MSC beneficial effect on myocyte function, potentially identifying novel therapeutics. The project is designed to both frame the research within a clinical context and provide specialized training of a future clinician-scientist.